Process for making antimicrobial articles by reacting chitosan with amino-reactive polymer surfaces

ABSTRACT

This invention relates to a process for making antimicrobial articles comprising contacting a chitosan solution with a polymer surface that contains amino-reactive functional groups.

FIELD OF THE INVENTION

A process for rendering a polymer article antimicrobial comprisingcontacting a chitosan solution with a polymer surface that containsamino-reactive functional groups.

TECHNICAL BACKGROUND OF THE INVENTION

This invention relates to a process for rendering a polymer articleantimicrobial, comprising contacting a chitosan solution with thesurface of a polymer that contains amino-reactive functional groups aspolymerized.

As evidenced by the presence in the market of numerous materials foreliminating or minimizing human contact with bacteria, there is clearlya demand for materials and/or processes that either minimize or killbacteria encountered in the environment. Such materials are useful inareas of food preparation or handling and in areas of personal hygiene,such as bathrooms. Similarly, there is a use for such antibacterialmaterials in hospitals and nursing homes where people with loweredresistance are especially vulnerable to bacteria.

Chitosan is the commonly used name for poly-[1-4]-β-D-glucosamine.Chitosan is chemically derived from chitin, which is apoly-[1-4]-β-N-acetyl-D-glucosamine, which, in turn, is derived from thecell walls of fungi, the shells of insects and, especially, crustaceans.Thus, it is inexpensively derived from widely available materials. It isavailable as an article of commerce from, for example, BiopolymerEngineering, Inc. (St. Paul, Minn.); Biopolymer Technologies, Inc.(Westborough, Mass.); and CarboMer, Inc. (Westborough, Mass.).

Chitosan can be treated with metal salt solutions so that the metal ionforms a complex with the chitosan. Chitosan and chitosan-metal compoundsare known to provide antimicrobial activity as bacteriocides andfungicides (see, e.g., T. L. Vigo, “Antimicrobial Polymers and Fibers:Retrospective and Prospective,” in Bioactive Fibers and Polymers, J. V.Edwards and T. L. Vigo, eds., ACS Symposium Series 792, pp. 175-200,American Chemical Society, 2001). Chitosan is also known to impartantiviral activity, though the mechanism is not yet well understood(see, e.g., Chirkov, S. N., Applied Biochemistry and Microbiology(Translation of Prikladnaya Biokhimiya i Mikrobiologiya) (2002), 38(1),1-8). Additionally, chitosan is known to impart antiodor properties;see, for example, WO 1999061079(A1).

Japanese Kokai 05269181 discloses the preparation of antimicrobialpolymers for contact lenses and containers for contact lenses. Thereference discusses chitosan being reacted with the surface of anoptically clear contact lens material. Exemplified aremethacrylate/carbonate copolymers with hydroxyl functionality. In oneexample, chitosan is attached to the surface by graft polymerization incarbodiimide aqueous solution onto an acrylic acid layer that has beenfirst grafted onto the contact lens. In another example, a solution ofchitosan in N-methyl-pyrrolidone contacts the contact lens, and thechitosan is crosslinked.

U.S. Pat. No. 5,618,622 discloses a surface-modified fibrous filtrationmedium which includes hydrocarbon polymer fibers having cationic oranionic functional groups on the surfaces thereof, coated with apolyelectrolyte of opposite charge, such as chitosan. There is nomention of antimicrobial properties.

Japanese patent application JP01-0342435 discloses an antimicrobialcoating agent that includes both chitosan and an emulsion or aqueousdispersion of a synthetic resin selected from among copolymers whichinclude unsaturated carboxylic acids as monomer components and ionomersobtained by partially or totally neutralizing said copolymers with metalions. The chitosan is mixed at a ratio of about 15 to 70 parts by weightwith respect to 100 parts by weight of the aforementioned syntheticresin. The surface is thus a mixture of the chitosan and the syntheticpolymer. The degree of deacetylation of the chitosan is 40-55%. Itssolubility in water implies that its molecular weight is low, perhapsunder 10,000. This reference also teaches away from coating an acidifiedsolution of chitosan onto a film or sheet surface.

In co-pending U.S. patent application No. 2003/0091612, polyolefinarticles are treated with an aqueous mixture of chromic acid andsulfuric acid, washed with deionized water, soaked in concentratednitric acid, and again washed with deionized water before treatment withchitosan solution. While effective antimicrobial articles are made bythis method, a simpler, more economical process is desirable.

It is therefore an object of this invention to provide an antimicrobialpolymeric material and a process for producing same, said processcomprising contacting a chitosan solution with a polymer surface thatcontains amino-reactive functional groups. Also provided are articlescomprising such material.

SUMMARY OF THE INVENTION

The invention discloses an antimicrobial polymeric material comprising apolymer that contains amino-reactive functional groups as polymerizedand a chitosan coating wherein the chitosan is reacted with saidfunctional groups, so that the surface of the polymeric material has achitosan concentration of at least 1000 ppm by area.

Also disclosed are articles comprising said composition and a processfor preparing said articles, said process comprising the sequentialsteps of:

-   -   a) providing an article whose surface comprises a polymer that        inherently contains amino-reactive functional groups;    -   b) contacting the article with a solution comprising chitosan;    -   c) optionally, contacting the article of step b) with a solution        containing a metal salt; and    -   d) drying the-article produced in step b) or c).

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings consist of 7 figures as follows:

FIG. 1 is a diagram showing the antimicrobial effect ofchitosan-treated, maleic anhydride-grafted low density polyethylenebeads vs. E. coli ATCC 25922.

FIG. 2 is a diagram showing the antimicrobial effect ofchitosan-treated, maleic anhydride-grafted low density polyethylenebeads vs. E. coli O157:H7, with and without silver doping.

FIG. 3 is a diagram showing the antimicrobial effect ofchitosan-treated, maleic anhydride-grafted high density polyethylenebeads vs. E. coli O157:H7, with and without silver doping.

FIG. 4 is a diagram showing the antimicrobial effect ofchitosan-treated, maleic anhydride-grafted low density polyethylenefilms vs. E. coli ATCC 25922.

FIG. 5 is a diagram showing the antimicrobial effect of chitosan-treatedfilms of Surlyn® thermoplastic resin and Nucrel® ethylene acid copolymervs. E. coli ATCC 25922.

FIG. 6 is a diagram showing the antimicrobial effect of chitosan-treatedNafion® 117 film vs. E. coli ATCC 25922.

FIG. 7 is a diagram showing the antimicrobial effect of chitosan-treatedsulfonated polyester yarn vs. E. coli ATCC 25922.

DETAILED DESCRIPTION OF THE INVENTION

“Amino-reactive groups” as used herein refers to chemicalfunctionalities that readily undergo chemical reaction with an NH₂group. Examples include positively charged species such as metal ions,anhydrides, carboxylic acids, isocyanates, epoxides, acid chlorides, andenones.

The phrase “a polymer that contains amino-reactive functional groups aspolymerized” as used herein refers to homopolymers and copolymers(including graft copolymers) which, as (co)polymerized, present asurface containing amino-reactive functional groups in sufficientquantity that the amino groups of the chitosan agent react with thesubstrate's surface to form a stable coating with a surfaceconcentration of at least 1000 ppm chitosan, without the need foradditional chemical or physical modification or priming of thesubstrate's surface (for example, treatment with caustic, acid, orplasma etching). Such a surface concentration of chitosan is desired foradequate antimicrobial effectiveness.

Polymer blends comprising said homopolymers and/or (co)polymers may beused in the present invention as long as the blend meets the requirementthat the amino groups of the chitosan agent react with the substrate'ssurface to form a stable coating with a surface concentration of atleast 1000 ppm chitosan.

One polymer type suitable for the present invention includes graftcopolymers such as, but not limited to, those described in U.S. Pat. No.4,026,967, in which the graft monomers include thermally stableunsaturated carboxylic anhydrides and dianhydrides, and the backbonepolymers are preferably polymers of ethylene and copolymers derived fromethylene and C₃-C₈ alpha-olefins, including copolymers of at least oneolefin with other monomers. Examples of suitable graft monomers for usein the present invention include methacrylic acid, acrylic acid,glycidyl methacrylate, 2-hydroxy ethylacrylate, 2-hydroxy ethylmethacrylate, diethyl maleate, monoethyl maleate, di-n-butyl maleate,maleic anhydride, maleic acid, fumaric acid, itaconic acid, itaconicanhydride, dodecenyl succinic anhydride, 5-norbornene-2, 3-anhydride,and nadic anhydride (3,6-endomethylene-1,2,3,6-tetrahydrophthalicanhydride). Fumaric acid, maleic anhydride, and glycidyl methacrylateare particularly preferred graft monomers. Examples of suitable backbonepolymers are polypropylene; polyethylene, e.g., high densitypolyethylene (HDPE), low density polyethylene (LDPE), linear low densitypolyethylene (LLDPE), metallocene-catalyzed polyethylene, very lowdensity polyethylene (VLDPE), ultrahigh molecular weight polyethylene(UHMWPE), high performance polyethylene (HPPE); copolymers of ethyleneand propylene; copolymers derived from ethylene or propylene and atleast one monomer chosen from propylene, methyl acrylate, ethylacrylate, n-butyl acrylate, methyl methacrylate, acrylic acid,methacrylic acid and carbon monoxide; and copolymers of olefins with adiolefin, such as a copolymer of ethylene, or of propylene, or ofethylene and other olefins, with: linear aliphatic nonconjugated dienesof at least six carbon atoms (such as 1,4-hexadiene) and other dienes,conjugated or not, such as norbornadiene, dicyclopentadiene, ethylidenenorbornene, butadiene, and the like. Other suitable backbone polymersare copolymers of ethylene and tetrafluoroethylene, such as Tefzel® ETFEfluoropolymer resin available from E. I. du Pont de Nemours & Co., Inc.(Wilmington, Del.). One example of a commercially available graftcopolymer suitable for use in the present invention is Bynel® 4033, amaleic anhydride grafted HDPE available from E. I. du Pont de Nemours &Co., Inc. (Wilmington, Del.).

Another type of polymer suitable for use in the present invention is acopolymer of an olefin with an acrylic and/or methacrylic acid. Ethyleneis the preferred olefin. An example of a commercially available materialis Nucrel® ethylene acid copolymer resin available from E. I. du Pont deNemours & Co., Inc. (Wilmington, Del.).

Other polymers suitable for use in the present invention are ionomers.The term “ionomer” as used herein refers to a polymer with inorganicsalt groups attached to the polymer chain (Encyclopedia of PolymerScience and Technology, 2nd ed., H. F. Mark and J. I. Kroschwitz eds.,vol. 8, pp. 393-396). Two typical ionomer structures are shown below:

where the ratio of m to n is usually on the order of 10 to 100; that is,typically only about 1 to 9% of the repeat units contain ionic groups.Ions M are typically metal ions like lithium, sodium, lithium, or zincbut can be other cations, for example, ammonium. Typically, an acid formof the polymer is made first and then neutralized to the desired degreewith base containing the desired metal ions. Partially neutralizedpoly(ethylene-co-methacrylic acid) and partially neutralizedpoly(ethylene-co-acrylic acid) are examples of ionomers, as issulfonated polystyrene. Some examples of ionomers that have beencommercialized are Surlyn® thermoplastic resin available from E. I. duPont de Nemours & Co., Inc. (Wilmington, Del.); Nafion® perfluorinatedsulfonic acid membranes, also from DuPont; Flemion® perfluorocarboxylateionomers developed by Asahi Glass Company in Japan; and a sulfonatedethylene-propylene terpolymer from Exxon. Polyesters and polyamides thathave been polymerized with a low level of sulfonated comonomer toenhance textile dyeability (see, e.g., U.S. Pat. Nos. 5,559,205;5,607,765; and 3,389,549) and sulfonated aromatic polyamides (see, e.g.,U.S. Pat. Nos. 3,567,632 and 4,595,708) such as those used in reverseosmosis membranes and other selective separation membranes are alsosuitable substrates for the present invention.

Examples of suitable polymer blends for use in the present inventioninclude but are not limited to toughened grades of semicrystallinethermoplastics, such as toughened polyesters and polyamides, wherein thetoughener is a polymer that contains amino-reactive groups aspolymerized.

The present invention is directed to antimicrobial polymeric materialand articles comprising same. Articles prepared by the methods of theinvention exhibit antimicrobial functionality wherein microbial growthis reduced as the article is commonly used. The term “antimicrobial” asused herein, means bactericidal, fungicidal, and antiviral as iscommonly known in the art. By “antimicrobial growth is reduced” or“reduction of bacterial growth” is meant that a 99.9% kill of thebacteria in 24 hours has been met as measured by the Shake Flask testdescribed below and as is commonly used to measure antimicrobialfunctionality which indicates a minimum requirement of a 3-log reductionin bacterial growth.

The articles of the present invention have at least one layer ofchitosan reacted thereon. Chitosan is the commonly used name forpoly-[1-4]-β-D-glucosamine. Chitosan is chemically derived from chitin,which is a poly-[1-4]-β-N-acetyl-D-glucosamine that, in turn, is derivedfrom the cell walls of fungi, the shells of insects and, especially,crustaceans.

As an optional first step of the present invention, the outer surface ofthe article is cleaned using any method or cleaning agent commonly knownin the art for the specific polymer that the article comprises. Forexample, the surface of a polyolefin article can be cleaned with C₁ toC₆ alcohols, dialkyl formamide and acetamide or with other polarsolvents capable of extracting plasticizers. The surface of a cleanedarticle may then, if necessary, be dried by methods commonly known inthe art, for example, by vacuum, ambient air drying, oven drying, andair forced drying.

Following the optional surface cleaning step, the article is treatedwith chitosan. This comprises soaking or wetting the article with achitosan treating solution. Typically, this treating solution is anaqueous acetic acid solution, preferably about 0.5% to about 5% aqueousacetic acid. In a preferred embodiment, an aqueous solution containing0.1% to 3% chitosan and 0.5% to 1.0% acetic acid is prepared. In more apreferred embodiment, an aqueous solution containing 2% chitosan and0.75% acetic acid is prepared. In another preferred embodiment, 2%chitosan and 1.5% aqueous acetic acid solution is prepared. The time oftreatment is typically 5 to 30 minutes. The temperature of the treatmentis not critical and is typically in the range of room temperature to 90°C.

After treatment with chitosan, the article may be washed, preferablywith deionized water. Optionally, the article may then be dried viamethods known in the art. Such methods include ambient air drying, ovendrying, and air forced drying. An inert atmosphere, such as nitrogen,may be provided in place of air. In a preferred embodiment, the articlesare oven dried at about 40-90° C. for about 12 to 24 hours.

Articles prepared by the methods of the present invention exhibitantimicrobial properties and are expected to inhibit odor development aswell. Said antimicrobial properties may, optionally, be further enhancedby treatment with metal salts. Metal salts useful for the presentinvention include, for example, zinc sulfate, copper sulfate, silvernitrate, or other water-soluble zinc, copper, and silver salts ormixtures of these. The metal salts are typically applied by dipping,spraying or padding a dilute (0.1% to 5%) solution of the salt in wateronto the article.

Articles comprising the polymeric material of the present invention maybe in the form of or comprise a film, membrane, laminate, knit fabric,woven fabric, nonwoven fabric, fiber, filament, yarn, pellet, coating,or foam. Articles may be prepared by any means known in the art, suchas, but not limited to, methods of injection molding, extruding, blowmolding, thermoforming, solution casting, film blowing, knitting,weaving, or spinning.

The preferred articles of the present invention provide multiple uses,since many articles benefit from a reduction in microbial growth and awide variety of polymers are included in the present invention. Thefollowing are examples of articles wherein it is desirable to reducemicrobial growth in or on the article in the end-use for which theparticular article is commonly used.

The articles of the invention include packaging for food, personal care(health and hygiene) items, and cosmetics. By “packaging” is meanteither an entire package or a component of a package. Examples ofpackaging components include, but are not limited, to packaging film,liners, absorbent pads packaging, shrink bags, shrink wrap, trays,tray/container assemblies, caps, adhesives, lids, and applicators. Suchabsorbent pads, shrink bags, shrink wrap, and trays of the presentinvention are particularly useful for packaging meat, poultry, and fish.

The package may be in any form appropriate for the particularapplication, such as a can, box, bottle, jar, bag, cosmetics package, orclosed-ended tube. The packaging may be fashioned by any means known inthe art, such as by extrusion, coextrusion, thermoforming, injectionmolding, lamination, or blow molding.

Some specific examples of packaging include, but are not limited to,bottles, tips, applicators, and caps for prescription andnon-prescription capsules and pills; solutions, creams, lotions,powders, shampoos, conditioners, deodorants, antiperspirants, andsuspensions for eye, ear, nose, throat, vaginal, urinary tract, rectal,skin, and hair contact; lip product packaging; and caps.

Examples of applicators include lipstick, chapstick, and gloss; packagesand applicators for eye cosmetics, such as mascara, eyeliner, shadow,dusting powder, bath powder, blusher, foundation and creams; and pumpdispensers and components thereof. These applicators are used to applysubstances onto the various surfaces of the body, and reduction ofbacterial growth will be beneficial in such applications.

Other forms of packaging components included in the present inventioninclude drink bottle necks, replaceable caps, non-replaceable caps, anddispensing systems; food and beverage delivery systems; baby bottlenipples and caps; and pacifiers. Where a liquid, solution or suspensionis intended to be applied, the package may be fashioned for applicationin a form for dispensing discrete drops or for spraying of droplets. Theinvention will also find use in pharmaceutical applications fashioned asinhalers.

Examples of end-use applications, other than packaging, in the area offood handling and processing that benefit from antimicrobialfunctionality and wherein microbial growth is reduced in the particularend-use of the consumer are coatings for components of food handling andprocessing equipment, such as temporary or permanent food preparationsurfaces; conveyer belt assemblies and their components; equipment formixing, grinding, crushing, rolling, pelletizing, and extruding andcomponents thereof; heat exchangers and their components; drains andtheir components; equipment for transporting water such as, but notlimited to, buckets, tanks, pipes, and tubing; and machines for foodcutting and slicing and components thereof. Where the surface of suchequipment components is metal, a coating of a polymer containingamino-reactive groups as polymerized could first be applied to the metalsurface. Alternatively, a film of such a polymer could be treated withchitosan and then heat sealed to the equipment surface. In oneembodiment, the equipment component is a screw for mixing and/orconveying that is an element in a single-screw or twin-screw extruder,such as, but not limited to, an extruder used for food processing; andthe polymer coating comprises an ionomer.

Articles of the present invention can also be used in or as items ofapparel, such as a swimsuit, undergarment, shoe component (for example,a woven or nonwoven shoe liner or insert), protective sports pad,child's garment, or medical garment (such as a gown, mask, glove,slipper, bootie, or head covering). Such garments particularly benefitfrom the inhibition of odor development.

Articles of the present invention can also be used in or as medicalmaterials, devices, or implants, such as bandages, adhesives, gauzestrips, gauze pads, medical or surgical drapes, syringe holders,catheters, sutures, IV tubing, IV bags, stents, guide wires, prostheses,orthopedic pins, dental materials, pacemakers, heart valves, artificialhearts, knee and hip joint implants, bone cements, vascular grafts,urinary catheter ostomy ports, orthopedic fixtures, pacemaker leads,defibrillator leads, ear canal shunts, cosmetic implants, ENT (ear,nose, throat) implants, staples, implantable pumps, hernia patches,plates, screws, blood bags, external blood pumps, fluid administrationsystems, heart-lung machines, dialysis equipment, artificial skin,ventricular assist devices, hearing aids, and dental implants.

In the hygiene area, articles of the present invention include personalhygiene garments such as diapers, incontinence pads, panty liners,sanitary napkins, sports pads, tampons and their applicators; and healthcare materials such as antimicrobial wipes, baby wipes, personalcleansing wipes, cosmetic wipes, diapers, medicated wipes or pads (forexample, medicated wipes or pads that contain an antibiotic, amedication to treat acne, a medication to treat hemorrhoids, ananti-itch medication, an anti-inflammatory medication, or anantiseptic).

Articles of the present invention also include items intended for oralcontact, such as a baby bottle nipple, pacifier, orthodontic applianceor elastic bands for same, denture material, cup, drinking glass,toothbrush, or teething toy.

Additional child-oriented articles that benefit through comprising thepolymeric material of the present invention include baby bottles, babybooks, plastic scissors, toys, diaper pails, and a container to holdcleansing wipes.

Household articles of the present invention include telephones andcellular phones; fiberfill, bedding, bed linens, window treatments,carpet, flooring components, foam padding such as mat and rug backings,upholstery components (including foam padding), nonwoven dryer sheets,laundry softener containing sheets, automotive wipes, household cleaningwipes, counter wipes, shower curtains, shower curtain liners, towels,washcloths, dust cloths, mops, table cloths, walls, and countersurfaces.

The current invention is also useful in reducing or preventing biofilmgrowth on the surface of selective separation membranes (for example,pervaporation, dialysis, reverse osmosis, ultrafiltration, andmicrofiltration membranes), and air and water filters that comprisepolymer with amino-reactive groups, for example, sulfonated aromaticpolyamides.

The current invention is also useful in providing an antifouling surfaceon boat components such as, but not limited to, boat hulls andcomponents thereof, and boat motors and components thereof. If thesurface of the boat component does not comprise a polymer withamino-reactive groups as polymerized, for example, if the boat componenthad a metal surface, a coating of a polymer containing amino-reactivegroups as polymerized could first be applied to the boat component'ssurface. Alternatively, a film of such polymer could be treated withchitosan and then heat sealed to the boat component's surface.

Devices used in fluid, e.g., water, transportation and/or storage canalso benefit from the antimicrobial polymeric material of the invention.Exemplary devices include, but are not limited to, pipes and tanks. Theinner surface, outer surface, or both surfaces of a pipe or tank cancomprise an antifouling surface of the invention. If the surface(s) doesnot comprise a polymer with amino-reactive groups as polymerized, forexample, if the surface(s) had a metal surface, a coating of a polymercontaining amino-reactive groups as polymerized could first be appliedto the surface(s). Alternatively, a film of such polymer could betreated with chitosan and then heat sealed to the surface(s).

In order to impart antimicrobial functionality to the products listed,the product can be treated with a chitosan agent according to the methodof the invention before it is manufactured, or after, or at any timeduring manufacture of the product. For example, in making anantimicrobial shower curtain, material having a surface that comprisesan effective amount of amino-reactive polymer can be treated accordingto the method of the invention, followed by fashioning a shower curtainfrom the treated material. Alternatively, the chitosan treatment may beperformed after the material is made into a shower curtain. It isbelieved that the antimicrobial properties of the material will notchange significantly.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and, withoutdeparting from the spirit and scope thereof, can make changes andmodifications to adapt the invention to various usages and conditions.

Materials and Methods:

The chitosan used in this study was material commercially availableunder the registered trademark ChitoClear® from Primex Corporation ofNorway. The material was used as purchased.

The degree of N-deacetylation of the chitosan samples was ascertained byproton and carbon 13 NMR spectroscopy to be over 85%. The molecularweight of the samples ranged from approximately 70,000 to approximately350,000.

Treated articles were tested for antimicrobial properties by the ShakeFlask Test for Antimicrobial Testing of Materials using the followingprocedure:

1. Inoculate a single, isolated colony from a bacterial or yeast agarplate culture in 15-25 ml of Trypticase Soy Broth (TSB) in a sterileflask. Incubate at 25-37° C. (use optimal growth temperature forspecific microbe) for 16-24 h with or without shaking (selectappropriate aeration of specific strain). For filamentous fungi, preparesporulating cultures on agar plates.

2. Dilute the overnight bacterial or yeast culture into sterilephosphate buffer (see below) at pH 6.0 to 7.0 to obtain approximately10⁵ colony forming units per ml (cfu/ml). The total volume of phosphatebuffer needed will be 50 ml×number of test flasks (including controls).For filamentous fungi, prepare spore suspensions at 10⁵ spores/ml. Sporesuspensions are prepared by gently resuspending spores from an agarplate culture that has been flooded with sterile saline or phosphatebuffer. To obtain initial inoculum counts, plate final dilutions(prepared in phosphate buffer) of 10⁻⁴ and 10⁻³ onto Trypticase Soy Agar(TSA) plates in duplicate. Incubate plates at 25-37° C. overnight.

3. Transfer 50 ml of inoculated phosphate buffer into each sterile testflask containing 0.5 g of material to be tested. Also, prepare controlflasks of inoculated phosphate buffer and uninoculated phosphate bufferwith no test materials.

4. Place all flasks on a wrist-action shaker and incubate with vigorousshaking at room temperature. Sample all flasks periodically and plateappropriate dilutions onto TSA plates. Incubate at 25 to 37° C. for 16to 48 h and count colonies.

5. Report colony counts as the number of Colony Forming Units per ml(cfu/ml).

6. The Δt value may be calculated as follows: Δt=C−B, where Δt is theactivity constant for contact time t, C is the mean log₁₀ density ofmicrobes in flasks of untreated control materials after X hours ofincubation, and B is the mean log₁₀ density of microbes in flasks oftreated materials after X hours of incubation. Δt is typicallycalculated at 4, 6, or 24 hours and may be expressed as Δt_(X).

Stock phosphate buffer: Monobasic Potassium Phosphate: 22.4 g DibasicPotassium Phosphate: 56.0 g Deionized Water: Bring up volume to 1000 ml

Adjust the pH of the phosphate buffer to pH 6.0 to 7.0 with either NaOHor HCl, filter, sterilize, and store at 4° C. until use. The workingphosphate buffer is prepared by diluting 1 ml of stock phosphate bufferin 800 ml of sterile deionized water.

Example 1 Preparation and antimicrobial evaluation of chitosan andchitosan-silver treated maleic anhydride-grafted polyethylene beads.

Low density polyethylene beads grafted with maleic anhydride (50 g,metallocene-catalyzed VLDPE, 2 Ml, 0.5% maleic anhydride graft) wereheated with 2% chitosan (Primex ChitoClear TM 1111, m.wt. 350,000)solution in 0.75% aqueous acetic acid (200 ml) at 80° C. for 30 minutes,cooled, filtered, and washed with deionized water. It was then dried at80° C. for 16 h. (Sample 1B).

Sample 1B (2 g) was immersed in 2% aqueous silver nitrate solution (10ml) and gently shaken for 30 min. The beads were then filtered andwashed three times with deionized water and dried under nitrogen at 40°C. (Sample 1C). These beads (1B and 1C) were evaluated for theirantibacterial properties against E. coli ATCC 25922, where ATTC refersto the American Type Culture Collection, and E. coli O157:H7, comparedwith untreated polymer beads (Sample 1A) and, as a positive control, achitosan-treated poly(ethylene terephthalate) fabric. This fabric wassequentially treated with 3% aqueous sodium hydroxide at 93° C. for 30min., neutralized with acetic acid, acidified with aqueous hydrochloricacid (pH 1.0), washed with water, and finally treated with chitosansolution (chitosan (Primex ChitoClear TM 588, m. wt, about 70,000, 2% in1% aqueous acetic acid solution), followed by drying the fabric at 77°C. for 8 h.

Samples of 1A, 1B, and 1C were also autoclaved with steam at 121° C. for20 minutes to sterilize them. The sterilized beads were then evaluatedfor antibacterial effectiveness. Results are shown in FIGS. 1 and 2.Untreated beads were not effective, chitosan treated ones were veryslightly effective, and silver plus chitosan treated, most effective.Antibacterial effectiveness remained after sterilization.

Example 2 Preparation and antimicrobial evaluation of antimicrobialmaleic anhydride-grafted high density Polyethylene beads

High density polyethylene beads grafted with maleic anhydride (50 g,Bynel® 4033) were heated with 2% chitosan (Primex ChitoClear® TM 1111,m.wt. 350,000) solution in 0.75% aqueous acetic acid (200 ml) at 80° C.for 30 minutes, cooled, filtered, and washed with deionized water. Itwas then dried at 80° C. for 16 h. (Sample 2A).

Sample 2A (10 g) was immersed in 2% aqueous silver nitrate solution (50ml) and gently shaken for 30 min. The beads were then filtered andwashed three times with deionized water and dried under nitrogen at 40°C. (Sample 2B). Samples 2A, 2B, and untreated beads (2C) were evaluatedfor their antibacterial properties against E. coli O157:H7. Beads of 2A,2B, and 2C that had been sterilized by autoclaving at 125° C. were alsoevaluated for antimicrobial activity. Results are shown in FIG. 3.Silver/chitosan treatment was most effective, and the antimicrobialactivity of the silver/chitosan-treated beads was retained aftersterilization.

Example 3 Preparation and antimicrobial evaluation of chitosan-treatedmaleic anhydride-grafted polyethylene films

Polyethylene film grafted on one side with maleic anhydride at a levelof 1 wt % was soaked in a solution of 2% chitosan (Primex ChitoClear® TM588, m.wt. 70,000) in aqueous 1.5% acetic acid for 30 min, air dried,then cured at 60° C. under nitrogen for 16 h. Three pieces of the film,samples A, B, and C, were evaluated for antimicrobial efficacy againstE. coli ATCC 25922 in a shake flask test. Results are shown in FIG. 4.

Example 4 Preparation of Nucrel®/Chitosan and Surlyn®/Chitosan films andantibacterial evaluation

Two film samples (A, B) each of Nucrel® 0403 ethylene-methacrylic acidcopolymer (typical methacrylic acid content of 4.0%), Nucrel® 0903ethylene-methacrylic acid copolymer (typical methacrylic acid content of9.0%), Surlyn® 1601 packaging resin (sodium ionomer, typical melt flowindex 1.3 dg/min by ASTM D1238, condition 190° C./2.16 kg), and Surlyn®1702 packaging resin (zinc ionomer, melt flow index 14.0 dg/min. by ASTMD1238, condition 190° C./2.16 kg) were weighed, soaked in 1 M aqueoushydrochloric acid for 30 min., washed with water, and then soaked in 2%chitosan solution (Primex ChitoClear® TM 656 in 0.5% aqueous aceticacid) overnight. The films were removed, the excess chitosan was allowedto drip off, and finally the films were dried at 60° C. under nitrogenatmosphere for 48 h. and re-weighed, as indicated in Table 1. It isthought that the few zero or negative weight gains are due toexperimental uncertainty and/or dehydration of an initially hydratedsample by the chitosan treatment. TABLE 1 Weight After Initial FilmChitosan Weight Sample Weight (g) Treatment (g) gain (g) Surlyn ® 1601(A) 5.90 5.96 0.06 Surlyn ® 1601 (B) 5.80 5.87 0.07 Surlyn ® 1702 (A)5.63 5.62 −0.01 Surlyn ® 1702 (B) 5.62 5.62 0.00 Nucrel ® 0903 (A) 5.845.90 0.06 Nucrel ® 0903 (B) 5.85 5.91 0.06 Nucrel ® 0403 (A) 2.94 2.980.04 Nucrel ® 0403 (B) 2.94 2.86 −0.08

The chitosan-treated B films were tested for antimicrobial propertiesagainst E. coli ATCC 25922 as described above, along with thecorresponding untreated films as controls. Results are shown in FIG. 5.

Example 5 Preparation of Nafion®-Chitosan film and antibacterialevaluation

A piece of Nafion® 117 perfluorosulfonic acid polymer film (about 183microns thick, weighing 10.91 g) was soaked in 2% chitosan solution(ChitoClear®, Primex TM 588, m.wt. about 70,000, in 1.5% aqueous aceticacid) for 5 min, air dried, followed by drying under nitrogen at 80° C.Weight of the dried film was 10.91 g. The lack of a net weight gain wasmost likely due to dehydration of the initially highly hydrated Nafion®117 film by the chitosan treatment. This was evaluated for antibacterialactivity against E. coli ATCC 25922 as described above, as was a controlfilm of untreated Nafion® 117 film. Results are shown in FIG. 6. Thechitosan-treated Nafion® 117 film provided a 2.6 log reduction of E.coli ATCC 25922 population after 2 hours, a 3.2 log reduction after 4hours, a 4.0 log reduction after 6 hours, and a 5.6 log reduction after8 hours. In contrast, the untreated Nafion® 117 film did not demonstrateantimicrobial activity.

Example 6 Preparation of antimicrobial sulfonated polyester yarn andantibacterial evaluation

Yarn made from poly(ethylene terephthalate) that had been copolymerizedwith a lithium salt of a glycollate of 5-sulfo-isophthalic acid at of alevel of approximately 1.75 mol % based on total diacids was passedsequentially through a tray containing water and a tray containing 2%chitosan solution (Primex ChitoClear® TM 588, m.wt. about 70,000, in1.5% aqueous acetic acid). The excess solution was stripped off, and theyarn was dried with a roller heated to 200° C. The wound yarn wassubsequently dried at 80° C. for a few days. Two samples (replicates)were taken and submitted for antimicrobial activity evaluation. Bothdemonstrated antimicrobial activity against E. coli ATCC 25922, as shownin FIG. 7.

1. A polymeric material comprising a) a polymer that containsamino-reactive functional groups as polymerized, and b) a chitosancoating wherein the chitosan is reacted with said functional groups,wherein the surface of said polymeric material has a chitosanconcentration of at least 1000 ppm by area.
 2. The polymeric material ofclaim 1, wherein the amino-reactive functional groups are metal ions,ammonium ions, anhydrides, carboxylic acids, sulfonic acid, isocyanates,epoxides, acid chlorides, enones, and combinations thereof.
 3. Thepolymeric material of claim 1, wherein the polymer is a homopolymer,random copolymer, block copolymer, graft copolymer, or polymer blend, atleast one monomer or comonomer of which contains at least one functionalgroup selected from the group consisting of metal ions, ammonium ion,anhydrides, carboxylic acid or carbonate, sulfonic acid or sulfonate,isocyanates, epoxides, acid chlorides, and enones.
 4. The polymericmaterial of claim 1, wherein the polymer is a graft copolymer comprisinga graft monomer that is a thermally stable unsaturated carboxylicanhydride or dianhydride, and a backbone polymer that is a homopolymerof ethylene, a homopolymer of propylene, a copolymer derived fromethylene and one or more C₃-C₈ alpha-olefins, or a copolymer derivedfrom propylene and one or more C₄-C₈ alpha-olefins.
 5. The polymericmaterial of claim 4, wherein the graft monomer is selected from thegroup consisting of methacrylic acid, acrylic acid, glycidylmethacrylate, 2-hydroxy ethylacrylate, 2-hydroxy ethyl methacrylate,diethyl maleate, monoethyl maleate, di-n-butyl maleate, maleicanhydride, maleic acid, fumaric acid, itaconic acid, itaconic anhydride,dodecenyl succinic anhydride, 5-norbornene-2,3-anhydride, and nadicanhydride (3,6-endomethylene-1,2,3,6-tetrahydrophthalic an hydride). 6.The polymeric material of claim 4, wherein the backbone polymer isselected from the group consisting of: a) polypropylene; b) polyethyleneselected from high density polyethylene, low density polyethylene,linear low density polyethylene, metallocene catalyzed polyethylene,very low density polyethylene, ultrahigh molecular weight polyethylene,and high performance polyethylene; c) copolymers of ethylene andpropylene; d) copolymers derived from ethylene or propylene and at leastone monomer selected from methyl acrylate, ethyl acrylate, n-butylacrylate, methyl methacrylate, acrylic acid, methacrylic acid, andcarbon monoxide; e) copolymers of olefins with a diolefin, wherein theolefins are selected from ethylene, propylene, and ethylene with otherolefins; and f) copolymers of ethylene and tetrafluoroethylene.
 7. Thepolymeric material of claim 6, wherein the diolefin is selected fromlinear aliphatic nonconjugated dienes of at least six carbon atoms,norbornadiene, dicyclopentadiene, ethylidene norbornene, and butadiene.8. The polymeric material of claim 1, wherein the polymer materialcomprises an ionomer.
 9. The polymeric material of claim 8, wherein theionomer is an ionomer of ethylene/acrylic acid copolymer or ofethylene/methacrylic acid copolymer; a perfluorinated sulfonate orcarboxylate polymer; a sulfonated polystyrene; a sulfonatedethylene-propylene terpolymer; a sulfonated polyester; or a sulfonatedpolyamide.
 10. The polymeric material of claim 1, wherein the polymer isa copolymer of ethylene with acrylic acid or methacrylic acid.
 11. Thepolymeric material of claim 1, further comprising one or more compoundsselected from metal salts, carboxyl-containing polymers, andcombinations thereof.
 12. The polymeric material of claim 11, whereinthe metal salts are selected from water-soluble zinc salt, water-solublecopper salt, water-soluble silver salt, and mixtures thereof.
 13. Anarticle comprising the polymeric material of claim
 1. 14. The article ofclaim 13 in the form of a film, membrane, laminate, knit fabric, wovenfabric, nonwoven fabric, fiber, filament, yarn, pellet, coating, orfoam.
 15. The article of claim 13, which has been blown, solution cast,laminated, injection molded, extruded, blow molded, thermoformed, knit,woven, or spun.
 16. The article of claim 13, wherein the article is apackage, packaging component, food or beverage dispensing system, babybottle, baby book, plastic scissors, toy, diaper pail, container forcleansing wipes, baby bottle nipple, pacifier, orthodontic appliance orcomponent thereof, denture material, cup, drinking glass, toothbrush,teething toy, tampon, tampon applicator, personal cleansing wipe, babywipe, cosmetic wipe, personal hygiene garment, food handling andprocessing equipment, item of apparel, household article, bandage,adhesive, gauze strip, gauze pad, medical or surgical drape, medicaldevice or implant, separation membrane, air or water filter, boatcomponent, or fluid transportation or storage device.
 17. The article ofclaim 16, wherein said package is a bottle, box, jar, can, bag,close-ended tube, cosmetics package, or inhaler.
 18. The article ofclaim 17, wherein said package contains a cosmetic, a personal hygienematerial, a healthcare material, or a combination thereof.
 19. Thearticle of claim 18, wherein said cosmetic, said personal hygienematerial or said healthcare material is lipstick, chapstick, eye shadow,eyeliner, mascara, dusting powder, bath powder, blusher, foundation,shampoo, conditioner, deodorant, antiperspirant, lotion, cream, powder,liquid, solution, suspension, capsule, or pill.
 20. The article of claim16, wherein said packaging component is in the form of a liner, lid,adhesive, replaceable or disposable container cap, film, shrink wrap,shrink bag, tray, tray/container assembly, absorbent pad for packaging,applicator, drink bottle neck, food dispensing system, or beveragedispensing system.
 21. Packaging for meat, poultry, or fish comprisingthe shrink wrap, shrink bag, tray, absorbent pad for packaging, andcombinations thereof of claim
 20. 22. The article of claim 16, whereinsaid applicator is a pump dispenser or component thereof, mascara wand,medicated pad or wipe, cosmetics brush, dropper, tip, lipstickapplicator, eyeliner applicator, or eye shadow applicator.
 23. Thearticle of claim 22, wherein said medicated pad or wipe comprises anantibiotic, a medication to treat acne, a medication to treathemorrhoids, an anti-itch medication, an anti-inflammatory medication,or an antiseptic.
 24. The article of claim 16, wherein said packagecontains a food or a beverage.
 25. The article of claim 16, wherein saidpersonal hygiene garment is a diaper, incontinence garment, panty liner,sanitary napkin, or tampon.
 26. The article of claim 16, wherein saidfood handling and processing equipment is selected from a conveyor beltassembly and components thereof; temporary and permanent foodpreparation surfaces; equipment for mixing, grinding, crushing, rolling,pelletizing, and extruding and components thereof; heat exchangers andtheir components; drains and their components; buckets, tanks, pipes,and tubing; and machines for food cutting and slicing and componentsthereof.
 27. The article of claim 26, wherein said equipment forextruding comprises a screw for mixing and/or conveying and wherein thepolymer coating comprises an ionomer.
 28. The article of claim 16,wherein said item of apparel is in the form of a swimsuit, sportswear,active wear, protective sports pad, undergarment, shoe component,child's garment, or medical garment.
 29. The article of claim 28,wherein said shoe component is a woven or nonwoven liner or insert. 30.The article of claim 28, wherein said medical garment is a gown, mask,glove, slipper, bootie, or head covering.
 31. The article of claim 16,wherein said household article is selected from the group consisting offiberfill; bedding; bed linens; window treatments; carpet and flooringcomponents; upholstery components; sheets; automotive wipes; nonwovendryer sheets; laundry softener-containing sheets; household cleaningwipes; counter wipes; towels; washcloths; dust cloths; mops;tablecloths; shower curtains; telephones and cellular phones; and wall,counter, and floor surfaces.
 32. The article of claim 16, wherein saidmedical device or implant is selected from the group consisting ofsyringe holders, catheters, sutures, IV tubing, IV bags, stents, guidewires, prostheses, orthopedic pins, dental materials, pacemakers, heartvalves, artificial hearts, knee and hip joint implants, bone cements,vascular grafts, bandages, adhesives, gauze strips, gauze pads, urinarycatheter ostomy ports, orthopedic fixtures, pacemaker leads,defibrillator leads, ear canal shunts, cosmetic implants, ENT implants,staples, implantable pumps, hernia patches, plates, screws, blood bags,external blood pumps, fluid administration systems, heart-lung machines,dialysis equipment, artificial skin, ventricular assist devices, hearingaids, and dental implants.
 33. The article of claim 16, wherein saidseparation membrane is a reverse osmosis, dialysis, pervaporation,ultrafiltration, or microfiltration membrane.
 34. The article of claim16, wherein said boat component is selected from the group consisting ofa boat hull, a component of a boat hull, a boat motor and a component ofa boat motor.
 35. The article of claim 16, wherein the fluidtransportation or storage device is a pipe or tank.
 36. A process forpreparing antimicrobial articles, said process comprising the sequentialsteps of: a) providing an article whose surface comprises a polymer thatcontains amino-reactive functional groups as polymerized; b) contactingthe article with a solution comprising chitosan; c) optionally,contacting the article of step b) with a solution containing a metalsalt; and d) drying the article produced in step b) or c).
 37. Theprocess according to claim 36, wherein the solution comprising chitosanof step b) also comprises 0.5% to 5% by volume aqueous acetic acid. 38.The process according to claim 36, wherein the solution comprisingchitosan of step b) comprises 0.5% to 1.0% by volume of aqueous aceticacid and 0.1% to 3% by volume of chitosan.
 39. The process according toclaim 36, wherein the metal salt of step d) is selected from the groupconsisting of water-soluble zinc salt, water-soluble copper salt,water-soluble silver salt, and mixtures thereof.